title: Engineering of cysteine residues leads to improved production of a human dipeptidase enzyme in E. coli.
abstract: Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human beta-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem beta-lactam antibiotics and can cleave dipeptides containing amino acids in both D: - and L: -configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.
authors.affiliation: Department of Chemical and Environmental Sciences and Materials and Surface Science Institute, University of Limerick, National Technology Park, Limerick, Ireland.
authors.forename: Ronan
authors.initials: R
authors.lastname: O'Dwyer
authors.forename: Rafia
authors.initials: R
authors.lastname: Razzaque
authors.forename: Xuejun
authors.initials: X
authors.lastname: Hu
authors.forename: Susan K
authors.initials: SK
authors.lastname: Hollingshead
authors.forename: J Gerard
authors.initials: JG
authors.lastname: Wall
mesh_terms: D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D004150:Dipeptidases; D003545:Cysteine